Evanescent hemolysis detection

ABSTRACT

Analyte content in a cell free portion of a body fluid, such as blood, is optically determined without centrifugation or other preliminary steps for separating the cell free portion from the body fluid. A channel is configured for containing a flowing sample of the body fluid along an optical boundary. The channel is configured so that a cell free layer of the fluid naturally forms along the boundary of the channel which coincides with the optical boundary. A light source is directed onto the optical boundary at an angle selected to generate total reflection from the boundary and to generate an evanescent field across the boundary in the cell free layer of fluid. A light detector is configured to detect absorption of the light in the evanescent field. The light source and light detector are matched to the wavelength range of an absorption peak of the analyte being detected.

RELATED APPLICATION

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 62/339,269, filed on May 20, 2016, the entirecontent of which is incorporated herein by reference.

FIELD OF TECHNOLOGY

Aspects of the present disclosure are directed to the field of clinicalanalyzers and more particularly to a method and apparatus for measuringfree hemoglobin in plasma without separating plasma from a whole bloodsample.

BACKGROUND

In a variety of clinical settings, it is important to measure certainchemical characteristics of plasma from whole-blood samples. Forexample, it is commonly important to measure the analytes, extracellularhemoglobin, bilirubin, and lipid particles in plasma. These settingsrange from a routine visit of a patient to a physician's office, anemergency room, or monitoring of a hospitalized patient, for example.Numerous techniques and apparatus are commonly used for measuringchemical characteristics of body fluids in clinical settings.Measurement of an analyte in a body fluid sample may be accomplished bynumerous methods one of which is by spectroscopic determination.

Some techniques for analyzing body fluid are complex and may involvenumerous steps such as centrifugation to prepare a fluid sample formeasurement. For example, techniques for measuring analyte content inthe plasma portion of a blood sample may involve preliminary steps suchas centrifugation of whole blood to separate blood cells from the plasmaportion. These preliminary steps add time, complexity and cost topreviously known techniques for measuring analyte content in a bodyfluid.

SUMMARY

The disclosed apparatus and method may be implemented to measureanalytes or components in the plasma fraction of a blood sample withoutany need for separation of plasma from the whole blood sample. Aspectsof the present disclosure provide a method and apparatus for quantifyinghemolysis in whole blood using frustrated total internal evanescent waveabsorption at a prism/blood interface. According to an aspect of thepresent disclosure, free hemoglobin in a whole blood sample can bemeasured using evanescent wave absorption without red blood cellseparation.

An apparatus for detecting analytes in whole blood without red bloodcell separation from the whole blood, the apparatus according to anaspect of the present disclosure includes a channel for receiving ablood sample, and a prism adjacent to the channel. A light sourcedirected through the prism at an angle of incidence greater than orequal to a critical angle relative to a normal of the interface, whereinthe angle of incidence creates total internal reflection of light fromthe first light source and creates an evanescent field extending intothe channel. The evanescent field decays to approximately zero withinabout 1 micron depth into the channel. When whole blood is flowing inthe channel, a substantially cell-free plasma layer occupies this thinboundary region of the channel. A light detector is aimed to receive thelight from the light source that has been reflected through the prismfrom an optical interface at the boundary of the channel. Analytecontent in a substantially cell-free plasma layer of the blood sample isdetermined by analysis of the reflected light. One aspect of the presentdisclosure describes an optical method for quantifying hemolysis inwhole blood using frustrated total internal reflection caused byevanescent wave absorption at a prism/blood interface.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the present disclosure, asillustrated in the accompanying drawings in which like referencecharacters refer to the same parts throughout the different views. Thedrawings, which are not necessarily to scale, emphasis illustrativeembodiments of the present disclosure.

FIG. 1 is an illustration of an apparatus for detecting analytes inwhole blood without red blood cell separation from the whole bloodaccording to an aspect of the present disclosure.

FIG. 2 is an illustration of an apparatus for detecting analytes inwhole blood without red blood cell separation from the whole bloodaccording to another aspect of the present disclosure.

FIG. 3 is an illustration of a prism integrated with a flow cell channelaccording to another aspect of the present disclosure

FIG. 4 is a graph showing light absorbance by a fluid sample having freehemoglobin versus wavelength of the light detected according to anaspect of the present disclosure.

FIG. 5 is a graph showing light absorbance for three samples havingdifferent levels of hemolysis versus wavelength of light detectedaccording to an aspect of the present disclosure.

FIG. 6 a graph of absorbance versus response time for six sample offluid having different levels of hemolysis as measured according to anaspect of the present disclosure.

FIG. 7 is a process flow diagram describing a method for detectinganalytes in whole blood without red blood cell separation from the wholeblood according to an aspect of the present disclosure.

DETAILED DESCRIPTION

When a whole blood sample flows through a channel having a small crosssectional diameter, such as a blood vessel in the body or a capillary ona chip, for example, the sample behaves as a flow stream in which asubstantially cell-free plasma film is present at the edges of thechannel. The substantially cell-free plasma film is a very thin layerhaving a thickness in the range of less than a micron to a few micronsat the edge of the channel. It is believed that the substantiallycell-free plasma film is present in blood vessels, for example, to helpprevent clogging and reduce fluidic resistance of the small bloodvessels in the body. The small blood vessels may have cross sectionaldiameter in a range of about 8 microns, for example.

According to aspects of the present disclosure, absorption of light ismeasured in the narrow substantially cell free plasma layer at theboundary of the flow channel and an optical interface. To measure theabsorption in this narrow region, light is incident onto the boundary atan angle greater than a critical angle. The incident light generates afield, called an evanescent wave, which penetrates into the flow cell.The optical field amplitude of the evanescent wave decays in less than 1wavelength, approximately 500 nm, from the flow cell surface. Becausethis optical path-length is so much smaller than typical co-oximetryflow cells (100 um), optical wavelengths corresponding to the maximumhemoglobin absorption, the Soret band around 420 nm, are used instead oftypical co-oximetry wavelengths in the range of 500-650 nm.

An evanescent field is an optical field that is created at the boundaryof two materials that have a different refractive index, e.g. between aglass prism, and a fluid like blood. The evanescent field exists onlynext to this interface and decays exponentially as you move away fromthe boundary. So, far away from the interface, the amplitude of thefield goes to zero. Because the evanescent field exists only next to theboundary, the plasma layer next to the boundary can be measured withoutthe field interacting with the cells.

According to an aspect of the present disclosure, the boundary layer isprobed with an evanescent field created by total internal reflectionfrom a prism surface. The presence of various analytes in plasma can bemeasured next to the channel wall without interference from the cellsbecause in the region very close to the wall the plasma is present withno cells.

An evanescent field is generated by configuring the angle of incidentlight with respect to an axis normal to the boundary to be greater thana certain critical angle by a margin of approximately 1-5 degrees. Thecritical angle depends on the nature of the two materials on either sideof the optical boundary. In an illustrative embodiment in which theoptical boundary is formed between a prism made from BK7 glass and bloodserum, for example, the critical angle is 62.4 degrees. When the angleof incidence is above the critical angle by a large enough margin, whichdepends on the light source being used, all of the incident light isreflected. That is called total internal reflection. Under conditions oftotal internal reflection, the only light on the other side of theboundary is called an evanescent field. On the other hand, when theangle of incidence is less than the critical angle, some of the incidentlight will propagate into the blood flow.

Because the evanescent light only penetrates a short distance into thechannel it provides only a weak absorption signal. Therefore, it isimportant that the light source emits light in a part of the spectrumthat provides good absorption by the analyte being detected. Anillustrative embodiment of the disclosed apparatus configured forhemolysis includes a light source that emits light in the 410 nm-420 nmwavelength range because in this range hemoglobin exhibits a very strongabsorption peak. In a particular embodiment, a light source that emitslight at 405 nm is used for hemolysis. In another embodiment in whichthe analyte being detected is bilirubin, a light source that emits lightwith a wavelength of 535 nm may be used. In still another embodiment inwhich the analyte being detected is lipemia, a light source that emitslight with a wavelength of 671 nm may be used.

According to an aspect of the present disclosure, two light sources maybe used for hemolysis. Differential detection may be performed bycomparing the absorption at the wavelength of a main signal withabsorption at some off-resonant wavelength. A first light sources mayprovide a main signal in the 420 nm wavelength range, for hemolysis. Thesecond light source may be provided in another color to correct forscattering and/or turbidity, or another absorbing analyte. Thewavelength of the second light source is not as critical as thewavelength of the first light source. In an illustrative embodiment, thesecond light source has a wavelength of about 470 nm. Because one or twocolors are used in certain embodiments of the disclosed apparatus, thelight detectors in these embodiments can be implemented as just onephotodiode for each color. It should be understood that the lightdetectors may alternatively be implemented as spectroscope inalternative embodiments. For example, an embodiment of the disclosedapparatus may be configured with light sources having numerous differentwavelengths. In these embodiments absorption may be measured using aspectroscope, for example.

Referring to FIG. 1, an apparatus 100 for detecting analytes in wholeblood without red blood cell separation from the whole blood accordingto an aspect of the present disclosure includes a channel 102 forreceiving a blood sample and a prism 104 adjacent to the channel 102.The prism 104 includes a first surface 106 abutting the channel 102 anddefining an optical interface 108 between the prism 104 and the bloodsample when the blood sample is received in the channel 102.

The apparatus 100 also includes a first light source 110 directedthrough the prism 104 to the optical interface 108 at an angle ofincidence 112 greater than or equal to a critical angle relative to anormal axis 114 of the interface. The angle of incidence 112 of opticalillumination in the prism 104 is greater than the critical angle of theprism/plasma interface 108. The angle of incidence 112 creates totalinternal reflection of light from the first light source 110 and createsan evanescent field 114 extending into the channel 102. The evanescentfield 114 extends into a plasma layer of the blood sample adjacent tothe interface 108 and decays to substantially zero before reaching aportion of the channel 102 containing blood cells.

In an embodiment according to another aspect of the present disclosure,the apparatus 100 may be configured for hemolysis detection in the wholeblood. In this embodiment the first light source 110 has an emissionwavelength in a range corresponding to a peak in an absorption spectraof hemoglobin. The emission wavelength of the first light source may bebetween about 410 nanometers and 420 nanometers, for example.

The apparatus 100 also includes a first light detector 116 aimed toreceive the light from the first light source 110 that has beenreflected through the prism 104 from the optical interface 108. Thefirst light source 110 may include a first light emitting diode and thefirst light detector 116 may include a first photodiode. In anotherillustrative embodiment, the first light detector 116 may include aspectroscope, for example.

Comparison circuitry coupled to the first light detector 116 isconfigured to identify a presence of analytes in the evanescent field114 by comparing intensity of the light that has been reflected throughthe prism 104 at a first wavelength with a predetermined intensity. Thepredetermined intensity may be an intensity of light emitted from thefirst light source 110, for example. The comparison circuitry mayinclude one or more processors coupled to computer memory, data storagedevices and/or communication circuitry and/or one or more computernetworks. For example, the comparison circuitry may and may includeconventional general purpose computer equipment or dedicated circuitryincorporated with an optical analysis module and configured formeasuring and/or comparing signals received by the first light detector.The comparison circuitry may also be configured to output and/or store ameasured level of analyte based on the measurements and/or comparisonsof the signals received by the first light detector, for example.

Referring to FIG. 2, an apparatus 200 for detecting analytes in wholeblood without red blood cell separation from the whole blood accordingto another aspect of the present disclosure includes a first lightsource 210 second light source 220 having an emission wavelengthdifferent than the emission wavelength of the first light source 210 anddirected through the prism 204 to the optical interface 208 at a secondangle of incidence greater than or equal to the critical angle relativeto the normal of the interface. The second angle of incidence createstotal internal reflection of light from the second light source 220 andcreates a second evanescent field extending into the channel 202. Inthis embodiment, the apparatus 200 also includes a second light detector226 coupled to the comparison circuitry and aimed to receive the lightfrom the second light source 220 that has been reflected through theprism 204 from the optical interface 208. In an illustrative embodiment,the comparison circuitry may be configured to compare the intensity ofthe light received by the first light detector 216 from the first lightsource 210 with an intensity of the light received by the second lightdetector 226 from the second light source 210.

In an illustrative embodiment, the flow cell 230 may be a conventionalflow cell bonded to a conventional prism 204, for example. The prism 204may have a rectangular face so that the flow cell 230 can be much longerthan the optical path-length through the prism 204. According to aspectsof the present disclosure, the prism 204 and/or the flow cell 230 may bemade from injection molded plastic or other inexpensive materials, forexample. In alternative embodiment according to an aspect of the presentdisclosure, the apparatus 200 may include a prism 204 in which thechannel 202 may be formed within the prism 204. Referring to FIG. 3, theprism 304 in this embodiment includes a flow cell channel 302 that hasbeen patterned into one face of the prism 304 to produce a measurementregion inside the prism 304.

An embodiment of the disclosed apparatus may configured as a simpledevice, having only one or two LEDs or laser diodes as light sources,one or two photo-diodes as light detectors, and a prism. The prism mayhave an integrated flow cell channel as shown in FIG. 3. In anillustrative embodiment, the entire apparatus could be configured in apackage having millimeter scale dimensions, for example.

FIG. 4 shows a graph 400 of light absorbance by a fluid sample having4.5 grams per deci-liter of free hemoglobin in units of milli-opticaldensity versus wavelength of the detected light. The graph 400 shows anabsorption peak 402 of hemoglobin in the blue 410 nm-420 nm portion ofthe optical spectrum, which is about ten times higher than minor peaks404 at about 540 nm and 406 at about 570 nm in the green portion of theoptical spectrum, and 100 times to 1000 times higher than peaks in thered portion of the optical spectrum. Configuring the light source with awavelength in the blue 410-420 nm range for hemolysis allows sufficientabsorption of the light by hemoglobin in the narrow cell-free boundaryof the channel and allows a good signal to noise ratio in the lightreceived by the light detector.

FIG. 5 shows a graph 500 of light absorbance for three samples havingdifferent levels of hemolysis in units of milli-optical density versuswavelength of the detected light. The graph 500 shows a first signal 502that shows no detectable peak for a sample having no hemolysis. A secondsignal 504 represents a sample having 2% hemolysis and has a peak ofabout 4 milli-optical density units in the 410 nm-420 nm range. A thirdsignal 506 represents a sample having 8% hemolysis and has a strong peakof about 18 milli-optical density units in the 410 nm-420 nm range. Thisshows that detection of absorbed light in the 410 nm-420 nm range by afluid sample is a strong indicator of an amount of an amount ofhemolysis in the sample.

FIG. 6 shows a graph 600 of absorbance in units of milli-optical densityversus response time in seconds for six sample of fluid having differentlevels of hemolysis. The graph 600 that at a time of (tm) of measurementthat is about 4 to 5 seconds after a measurement start time (t0) thereis a significant separation of signal levels representing absorption inthe 410-420 nm range for distinguishing different levels of hemolysis ina sample. For example at the time of measurement, a first signal 602representing a first sample having no hemolysis indicates no absorptionin the received light at wavelengths of 410 nm-420 nm. At the time ofmeasurement a second signal 604 representing a second sample having 50mg/dL of hemolysis indicates absorption of about 1 milli-optical densityunits in the received light at wavelengths of 410 nm-420 nm. At the timeof measurement a third signal 606 representing a third sample having 100mg/dL of hemolysis indicates absorption of about 1.5 milli-opticaldensity units in the received light at wavelengths of 410 nm-420 nm. Atthe time of measurement a fourth signal 608 representing a fourth samplehaving 200 mg/dL of hemolysis indicates absorption of about 2.5milli-optical density units in the received light at wavelengths of 410nm-420 nm. At the time of measurement a fifth signal 610 representing afifth sample having 400 mg/dL of hemolysis indicates absorption of about5 milli-optical density units in the received light at wavelengths of410 nm-420 nm. At the time of measurement a sixth signal 612representing a sixth sample having 800 mg/dL of hemolysis indicatesabsorption of about 11.5 milli-optical density units in the receivedlight at wavelengths of 410 nm-420 nm.

Referring to FIG. 7, another aspect of the present disclosure includes amethod 700 for detecting analytes in whole blood without red blood cellseparation from the whole blood. At block 702, the method 700 includesreceiving a whole blood sample in a channel. A prism adjacent to thechannel includes a first surface abutting the channel and defining anoptical interface between the prism and the blood sample when the wholeblood sample is received in the channel. At block 704, the method alsoincludes directing a first light source through a prism to the opticalinterface at an angle of incidence greater than or equal to a criticalangle relative to a normal of the interface. The angle of incidencecreates total internal reflection of light from the first light sourceand creates an evanescent field extending into the channel. At block706, the method also includes aiming a first light detector to receivethe light from the first light source that has been reflected throughthe prism from the optical interface. At block 708, the method includesmeasuring absorption of the light from the first light source by ananalytes in only a plasma layer of the whole blood sample within rangeof the evanescent field.

An apparatus for determining an analyte content in blood, according toanother aspect of the present disclosure includes an optical boundarybetween a flowing blood sample and an optically transmissive media, suchas a prism, for example. The apparatus includes an evanescent opticalfield in the flowing blood adjacent to the boundary, and a lightdetector such as a photo-diode or a spectroscope configured to detectabsorption of light in the evanescent field at a wavelengthcorresponding to an absorption wavelength of the analyte. According toanother aspect of the present disclosure, the apparatus also includes alight emitter, such as an light emitting diode or other light source,configured to direct light onto the optical boundary at a wavelengthcorresponding to the absorption wavelength of the analyte and at anangle of incidence selected to provide total internal reflection of thelight within the optically transmissive media. According to anotheraspect of the present disclosure, the apparatus also includes a channelcontaining the flowing blood, wherein the channel is configured togenerate a cell free layer of the flowing blood at a boundary of thechannel, and wherein the boundary of the channel comprises the opticalboundary.

In an illustrative embodiment the apparatus is configured fordetermining free hemoglobin content in the cell free layer of theflowing blood. In this embodiment, according to an aspect of the presentdisclosure, the light emitted by the light emitter has a wavelength ofbetween 410 nanometers and 420 nanometers, and the light detector isconfigured to detect light absorption at wavelengths between 410nanometers and 420 nanometers.

Although aspects of the present disclosure are described herein in thecontext of hemolysis, it should be understood by persons skilled in theart that aspects of the present disclosure can be implemented fordetecting various analytes and other constituents in a plasma fractionof body fluid sample.

What is claimed is:
 1. An apparatus for detecting analytes in wholeblood without red blood cell separation from the whole blood, theapparatus comprising: a channel for receiving a blood sample; a prismadjacent to the channel, wherein the prism includes a first surfaceabutting the channel, the first surface defining an optical interfacebetween the prism and the blood sample when the blood sample is receivedin the channel; a first light source directed through the prism to theoptical interface at an angle of incidence greater than or equal to acritical angle relative to a normal of the interface, wherein the angleof incidence creates total internal reflection of light from the firstlight source and creates an evanescent field extending into the channel,wherein the evanescent field extends into a plasma layer of the bloodsample adjacent to the interface and decays to substantially zero beforereaching a portion of the channel containing blood cells; and a firstlight detector aimed to receive the light from the first light sourcethat has been reflected through the prism from the optical interface. 2.The apparatus of claim 1, further comprising: comparison circuitrycoupled to the first light detector, wherein the comparison circuitry isconfigured to identify a presence of analytes in the evanescent field bycomparing intensity of the light that has been reflected through theprism at a first wavelength with a predetermined intensity.
 3. Theapparatus of claim 2, wherein the predetermined intensity is anintensity of light emitted from the first light source.
 4. The apparatusof claim 1, further comprising a second light source having an emissionwavelength different than the emission wavelength of the first lightsource and directed through the prism to the optical interface at asecond angle of incidence greater than or equal to the critical anglerelative to the normal of the interface, wherein the second angle ofincidence creates total internal reflection of light from the secondlight source and creates a second evanescent field extending into thechannel; and a second light detector coupled to the comparison circuitryand aimed to receive the light from the second light source that hasbeen reflected through the prism from the optical interface, wherein thecomparison circuitry is configured to compare the intensity of the lightreceived by the first light detector from the first light source with anintensity of the light received by the second light detector from thesecond light source.
 5. The apparatus of claim 1 configured forhemolysis detection in the whole blood, wherein the first light sourcehas an emission wavelength in a range corresponding to a peak in anabsorption spectra of hemoglobin.
 6. The apparatus of claim 5, whereinthe emission wavelength of the first light source is between about 410nanometers and 420 nanometers.
 7. The apparatus of claim 1 configuredfor detecting analytes in the whole blood, wherein the analytes areselected from the group consisting of hemoglobin, bilirubin and lipemia.8. The apparatus of claim 1, wherein the channel is formed within theprism.
 9. The apparatus of claim 1, wherein the first light sourcecomprises a first light emitting diode and wherein the first lightdetector comprises a first photodiode.
 10. The apparatus of claim 1,wherein the first light source comprises a laser diode.
 11. Theapparatus of claim 1, wherein the first light detector comprises aspectroscope.
 12. A method for detecting analytes in whole blood withoutred blood cell separation from the whole blood, the method comprising:receiving a whole blood sample in a channel, wherein a prism adjacent tothe channel includes a first surface abutting the channel, the firstsurface defining an optical interface between the prism and the bloodsample when the whole blood sample is received in the channel; directinga first light source through a prism to the optical interface at anangle of incidence greater than or equal to a critical angle relative toa normal of the interface, wherein the angle of incidence creates totalinternal reflection of light from the first light source and creates anevanescent field extending into the channel, wherein the evanescentfield extends into a plasma layer of the blood sample adjacent to theinterface and decays to substantially zero before reaching a portion ofthe channel containing blood cells; aiming a first light detector toreceive the light from the first light source that has been reflectedthrough the prism from the optical interface; and measuring absorptionof the light from the first light source by an analytes in only a plasmalayer of the whole blood sample within range of the evanescent field.13. An apparatus for determining an analyte content in blood, theapparatus comprising: an optical boundary between a flowing blood sampleand an optically transmissive media; an evanescent optical field in theflowing blood adjacent to the boundary, wherein the evanescent fieldextends into a plasma layer of the blood sample adjacent to theinterface and decays to substantially zero before reaching a portion ofthe channel containing blood cells; and a light detector configured todetect absorption of light in the evanescent field at a wavelengthcorresponding to an absorption wavelength of the analyte.
 14. Theapparatus of claim 13, wherein the light detector comprises aphoto-diode.
 15. The apparatus of claim 13, wherein the light emittercomprises a laser diode.
 16. The apparatus of claim 13, wherein theoptically transmissive media comprises a prism.
 17. The apparatus ofclaim 13, further comprising: a light emitter configured to direct lightonto the optical boundary at a wavelength corresponding to theabsorption wavelength of the analyte and at an angle of incidenceselected to provide total internal reflection of the light within theoptically transmissive media.
 18. The apparatus of claim 17, wherein thelight emitter comprise a light emitting diode.
 19. The apparatus ofclaim 17, wherein the light emitter comprises a laser diode.
 20. Theapparatus of claim 17, further comprising a channel containing theflowing blood, wherein the channel is configured to generate a cell freelayer of the of the flowing blood at a boundary of the channel, andwherein the boundary of the channel comprises the optical boundary. 21.The apparatus of claim 20, wherein the analyte comprises free hemoglobinin the cell free layer of the flowing blood.
 22. The apparatus of claim21, wherein the light emitted by the light emitter has a wavelength ofbetween 410 nanometers and 420 nanometers.
 23. The apparatus of claim22, wherein the light detector is configured to detect light absorptionat wavelengths between 410 nanometers and 420 nanometers.
 24. Theapparatus of claim 20, wherein the analyte is in the group consisting ofhemoglobin, bilirubin and lipemia.